Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases

Zaruhi Hovhannisyan, Jacquelyn Treatman, Dan R Littman, Lloyd Mayer, Zaruhi Hovhannisyan, Jacquelyn Treatman, Dan R Littman, Lloyd Mayer

Abstract

Background & aims: Interleukin (IL)-17-producing CD4(+) helper T cells (Th17) mediate mucosal immunity and are involved in the pathogenesis of inflammatory bowel disease (IBD). They are believed to arise from the same precursor population as regulatory T (Treg) cells, but little is known about how these T-cell subsets interact under chronic inflammatory conditions. We studied Th17 and Treg cells isolated from intestinal lamina propria of patients with IBD to investigate their role in pathogenesis.

Methods: FoxP3 expression (a marker of Treg cells) and IL-17 production were assessed in CD4(+) lamina propria lymphocytes isolated from IBD patients and healthy subjects. IL-17(+)FoxP3(+) and IL-17(+) CD4(+) T-cell clones were generated by limiting dilution. An in vitro suppression assay was performed to assess the functional capacity of derived T-cell clones.

Results: IL-17(+)FoxP3(+) T cells were identified in inflamed intestinal mucosa of patients with Crohn disease (CD), but not in patients with ulcerative colitis (UC) or healthy controls. These cells shared phenotypic characteristics of Th17 and Treg cells, and showed potent suppressor activity in vitro. Transforming growth factor-β was necessary and sufficient to induce development of an IL-17(+) FoxP3(+) cell population in CD4(+) lamina propria lymphocytes derived from patients with UC.

Conclusions: The inflammatory environment in the intestinal mucosa of patients with CD contributes to the generation of a distinct population of Treg cells that are FoxP3(+) and produce IL-17. These cells are likely to arise during differentiation of Th17 and Treg cells. Specific microenvironmental cues from tissues are likely to determine their commitment to either lineage and affect the balance between regulation and inflammation in the intestine.

Conflict of interest statement

Disclosures: The authors have no conflict of interests to disclose.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
A distinct population of FoxP3+ IL-17-producing CD4+ T cells is present in the inflamed intestinal mucosa of CD patients, but not UC patients or healthy controls. (A) Flow cytometry analyzing the expression of FoxP3 and IL-17 by lamina propria (LP) CD4+ T lymphocytes freshly derived from disease-affected and apparently healthy gut areas of IBD patients and healthy individuals, respectively. Numbers in the quadrants indicate the percent of cells in each. Data are representative of at least twenty independent experiments. (B) Flow cytometric analysis of the distribution of FoxP3+IL17+ CD4+ T cells within the bowel in CD patients and healthy controls. The frequencies of FoxP3+ IL17-producing T cells derived from inflamed CD tissues are indicated in the circle. (C) Summary figure: flow cytometric analysis of the frequencies of FoxP3+IL17+ CD4+ T cells in inflamed, slightly- or non-inflamed gut areas of CD patients and healthy gut areas of normal controls. Means (small horizontal bars): healthy controls (n=18), 0.3 (s.d., 0.2); inflamed gut areas from CD patients (n=27), 3.6 (s.d., 0.52); slightly or non-inflamed gut areas from CD patients (n=10), 1.2 (s.d., 0.32). **, P≤0.002. ***, P≤0.0001. Each symbol represents the percent of cells co-expressing IL-17 and FoxP3 for a single donor (B, C). (D) Comparative assessment of FoxP3+IL17+ cell frequencies in the peripheral blood (PB), inflamed gut and gut-draining mesenteric lymph node (MLN) compartments derived from the same CD patient (error bars, s.d.; n=10). **, P≤0.001. Intracellular staining for IL-17 production and FoxP3 expression was performed on sorted LP CD4+ T cells 4h post stimulation with PMA/ionomycin (A, B, C, D).
Figure 2
Figure 2
CD-derived FoxP3+ IL-17-producing CD4+ T cells display phenotypic similarities to both Th17 and Treg cells. (A, B, C) Surface and intracellular staining of CD-derived LP CD4+ T cells 4h post stimulation with PMA/ionomycin. (A) Surface expression of integrin α4β7, CCR6 and CD103 by LP FoxP3+IL-17+ cells. (B) Expression of RORγt, CD161, CD101 and CD127 by LP FoxP3+IL-17+ cells (C) Cytokine secretion profile of LP FoxP3+IL-17+cells. (A, B, C) LP CD4+ T cells were co-stained with anti-IL-17 and anti-FoxP3. Gating on FoxP3+IL-17+ cells: filled histograms, specific staining (antibodies to markers below the plots); open histograms, isotype-matched control antibodies. Numbers adjacent to outlined areas and above lines indicate the percent of cells in the gate. Data are representative of at least seven independent experiments.
Figure 3
Figure 3
TCR repertoire analysis of CD-derived FoxP3+ IL-17-producing LP CD4+ T cells. Sorted LP CD4+ T cells were stained with anti-IL-17 and anti-FoxP3 4h post stimulation with PMA/ionomycin. The TCR β-chain variable region usage of IL-17+, FoxP3+IL-17+ and FoxP3+ T cell subsets was defined by flow cytometry using specific anti-Vβ TCR antibodies. Mean values are shown for six different CD donors. *, P≤0.05. **, P≤0.01. ***, P≤0.001
Figure 4
Figure 4
CD-derived FoxP3+IL-17+ T cell clones suppress proliferation of responder CD4+ T cells. (A) In vitro suppression assay with CFSE-labeled PB-derived CD4+ T cells cultured with αCD3/αCD28 beads in the absence or presence of conventional CD4+CD25+ Tregs, CD-derived FoxP3+IL-17+ or IL-17+ T cell clones at effector/Treg ratios of 1:1. The proliferation of responder CD4+ T cells accompanied by CFSE dilution was analyzed by flow cytometry on day 4. Numbers above the lines indicate percent proliferating CD4+ T cells. (B) Suppression of responder CD4+ T cell proliferation in the presence of CD-derived FoxP3+IL-17+ T cell clones incubated at the indicated effector/Treg ratios. Data are representative of eight (A) or three (B) independent experiments.
Figure 5
Figure 5
TGF-β is necessary and sufficient for the induction of FoxP3+IL-17+ population in UC-derived LP CD4+ T cells. (A) Flow cytometry analyzing the expression of IL-17 and FoxP3 by LP CD4+ T cells derived from IBD patients and normal controls primed with αCD2/αCD3/αCD28 coated beads without cytokines or in the presence of various combinations of IL-1β, IL-6, IL-21, IL-23 and TGF-β (above the graph), with IL-2 added on day 3 and analyzed on day 6 of culture. Numbers in the quadrants indicate the percent of cells in each. (B) Flow cytometry analyzing the expression of IL-17 and FoxP3 by UC-derived LP CD4+ T cells primed with αCD2/αCD3/αCD28 coated beads in the absence or presence of various combinations of indicated cytokines and neutralizing anti-TGF-β (above the graph), with IL-2 added on day 3 and analyzed on day 6. Data are representative of four (A) and three (B) independent experiments.

References

    1. Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007;204:1849–1861.
    1. Bettelli E, Oukka M, Kuchroo VK. T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol. 2007;8:345–350.
    1. Aujila SJ, Dubin PJ, Kolls JK. Th17 cells and mucosal host defense. Semin Immunol. 2007;19:377–382.
    1. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature. 2006;441:235–238.
    1. Khader SA, Bell GK, Pearl JE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol. 2007;8:369–377.
    1. McKenzie BS, Kastelein RA, Cua DJ. Understanding the IL-21-IL-17 immune pathway. Trends Immunol. 2006;27:17–23.
    1. Ouyang W, Kools JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity. 2008;28:454–467.
    1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. FoxP3+ Treg cell functioning in suppression of effector T cells is essential in maintenance of peripheral tolerance. Cell. 2008;133:775–787.
    1. Zheng Y, Rudensky AY. FoxP3 in control of the regulatory T cell lineage. Nat Immunol. 2007;8:457–462.
    1. Wan Y, et al. TGF-β and regulatory T cell in immunity and autoimmunity. J Clin Immunol. 2008;28:647–659.
    1. Mangan PR, Harrington LE, O'Quinn DB, et al. Transforming growth factor-β induces development of the Th17 lineage. Nature. 2006;441:231–234.
    1. Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF-β1 maintains supressor function and FoxP3 expression in CD4+CD25+ regulatory T cells. J Epx Med. 2005;201
    1. Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcriptional factor FoxP3. J Epx Med. 2003;198:1875–1886.
    1. Yang XO, Nurieva R, Martinez GJ, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cells programs. Immunity. 2008;29:44–56.
    1. Zhou L, Lopes JE, Chong MM, et al. TGF-β-induced FoxP3 inhibits Th17 cell differentiation by antagonizing RORγt function. Nature. 2008;453:236–240.
    1. Koenen HJ, Smeets RL, Vink PM, et al. Human CD25high FoxP3+ regulatory T cells differentiate into IL-17-producing cells. Blood. 2008;112:2340–2352.
    1. Eberl G, Marmon S, Sunshine MJ, et al. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol. 2004;5:64–73.
    1. Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor ROR-gammat dirrects the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126:1121–1133.
    1. Voo KS, Wang YH, Santori FR, et al. Identification of IL-17-producing FoxP3+ regulatory T cells in humans. Proc Natl Acad Sci. 2009;106:4793–4798.
    1. Fujino S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003;52:65–70.
    1. Nielsen OH, Kirman I, Rüdiger N, et al. Upregulation of interleukin-12 and -17 in active inflammatory bowel disease. Scand J Gastroenterol. 2003;38:180–185.
    1. Seiderer J, Elben I, Diegelmann J, et al. Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL-17F p.His161Arg polymorphism in IBD. Inflamm Bowel Dis. 2008;14:437–445.
    1. Duerr RH, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006;314:1461–1463.
    1. Dubinsky MC, Wang D, Picornell Y, et al. IL-23 receptor (IL-23R) gene protects against pediatric Crohn's disease. Inflamm Bowel Dis. 2007;13:511–515.
    1. Maul J, Loddenkemper C, Mundt P, et al. Peripheral and intestinal regulatory CD4+CD25high T cells in inflammatory bowel disease. Gastroenterology. 2005;128:1868–1878.
    1. Saruta M, Yu QT, Fleshner PR, et al. Characterization of FoxP3+ CD4+ regulatory T cells in Crohn's disease. Clinical Immunology. 2007;125:281–290.
    1. Makita S, Kanai T, Oshima S, et al. CD4+CD25+ bright T cells in human intestinal lamina propria as regulatory cells. J Immunol. 2004;173:3119–3130.
    1. Liao Fea. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsivenedd to macrophage inflammatory protein 3 a. J Immunol. 1999;162:186–194.
    1. Kleinschek MA, Boniface K, Sadekova S, et al. Circulating and gut-resistant human Th17 cells express CD161 and promote intestinal inflammation. J Epx Med. 2009;206:525–534.
    1. Korn T, Bettelli E, Gao W, et al. IL-21 initiates an alternative pathway to induce proinflammatory T(H)-17 cells. Nature. 2007;448:484–487.
    1. Langrish CL, Chen Y, Blumenschein WM, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Epx Med. 2005;201:233–240.
    1. Nurieva R, Yang XO, Martinez G, et al. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature. 2007;448:480–483.
    1. Veldhoen M, Hocking RJ, Atkins CJ, et al. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24:179–189.
    1. Zhou L, Ivanov II, Spolski R, et al. IL-6 program T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol. 2007;8:967–974.
    1. Del Zotto B, Mumolo G, Pronio AM, et al. TGF-β1 production in inflammatory bowel disease:differing production patterns in Crohn's disease and ulcerative colitis. Clin Exp Immunol. 2003;134:120–126.
    1. León AJ, Gómez E, Garrote JA, et al. High levels of proinflammatory cytokines, but not markers of tissue injury, in unaffected intestinal areas from patients with IBD. Mediators Inflamm. 2009;2009:580450.

Source: PubMed

Sponsors et collaborateurs

Les conditions médicales

Interventions en matière de drogue

3
S'abonner